RESUMO
Meningococcal disease is a devastating infection caused by Neisseria meningitidis (meningococcus), and it is classified into serogroups according to its polysaccharide capsule composition. In Brazil, serogroup C is the most frequently responsible for the majority of cases, representing a serious public health challenge. In 2010, the meningococcal serogroup C conjugate vaccine was included in the calendar of the National Immunization Program. We have evaluated 163 meningococcal isolates collected during the pre (2006-2010) and post (2011-2016) vaccination periods. Epidemiological data were determined through Multilocus Sequence Typing (MLST) analysis, vaccine antigens and Bexsero Antigen Sequence Typing (BAST) variant. Clonal complex 103 remains the most prevalent in the country with a high number of serogroup C strains to which CC103 is directly associated. A total of 42 different ST were found. The two most prevalent ST were ST-3780 (CC103) with 38 strains and ST-10781, which was not associated with a CC with nine strains. Allele abcZ-276 was reported among 98% of the strains analyzed and it was not found among other CC103 strains worldwide, makes this allele an important genetic marker for a specific new clone only assigned to Brazilian serogroup C strains, ST-3780. FHbp-25 and NHBA-42 peptides were the most prevalent among isolates in both periods studied. BAST-824 and BAST-3073 have been expressed only in CC103 over the studied years, however, it was not possible to associate a BAST variant to a specific CC. Serogroup C phenotype [P1.22,14-6,36-2: F3-9: ST-3780 (CC103)] was the most prevalent according to the antigenic profiles of circulating strains in Brazil (2007-2016). Our study suggests that CC103 is still a major hypervirulent CC circulating in Brazil and ST-3780 is currently spreading all over the country even after the introduction of MenC in 2010.
Assuntos
Antígenos de Bactérias/genética , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Tipagem de Sequências Multilocus/métodos , Neisseria meningitidis Sorogrupo C/classificação , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias/imunologia , Brasil , Variação Genética , Humanos , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/genética , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo C/genética , Neisseria meningitidis Sorogrupo C/imunologia , Neisseria meningitidis Sorogrupo C/isolamento & purificação , Filogenia , Vigilância da População , SorogrupoRESUMO
Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.
Assuntos
Animais , Salmonella/isolamento & purificação , Contaminação de Alimentos , Separação Imunomagnética/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Microbiologia de Alimentos/métodos , Salmonella/genética , Proteínas de Bactérias/imunologia , Sensibilidade e Especificidade , Leite/microbiologia , Carne/microbiologia , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismoRESUMO
AIMS: To devise a protocol for heterologous expression and purification of a partial toxic portion of the Bacillus thuringiensis (Bt) vegetative insecticidal protein Vip3A and using it as an antigen for anti-Vip3A polyclonal antibody development. Also, to evaluate the regulation of Vip3A secretion into culture supernatants (SNs) of different Bt strains based on this antibody. METHODS AND RESULTS: A primer pair was designed to amplify partially the toxic portion of the vip3A gene from the HD125 strain. The amplicon was cloned in expressing vector to produce a ~35 kDa peptide, which was HPLC-purified prior to rabbit immunizations. The serum containing the polyclonal anti-Vip3A antibody demonstrated a detection sensitivity of 0·4 ng mm-2 for the antigen in slot-blot experiments. Seven Bt strains from different origins were assessed regarding their temporal secretion of Vip3A toxin. ELISA results showed a strain-specific temporal regulation of Vip3A secretion in culture for the temperate isolates, with no detection of the toxin for the tropical strains, even when the presence of the gene was confirmed by PCR and sequencing. CONCLUSIONS: Conformational variation in the toxic portion of Vip3A may explain lack of its detection in the tropical strains. Isolates from the same subspecies display physiological variability in proteins' secretion into culture SNs, which can affect screening procedures for more effective strains/toxins. SIGNIFICANCE AND IMPACT OF THE STUDY: Immunoassays based on the developed anti-Vip3A antibody can be useful in a variety of basic studies. This method can be also coupled with toxicity assays on target insects, for more efficient screening methods of novel Bt strains/toxins with biocontrol applicability.
Assuntos
Anticorpos Antibacterianos , Bacillus thuringiensis , Proteínas de Bactérias , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Inseticidas/imunologia , Inseticidas/metabolismo , CoelhosRESUMO
Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10h, which is a promising rapid method to detect Salmonella in emergency.
Assuntos
Contaminação de Alimentos , Microbiologia de Alimentos/métodos , Separação Imunomagnética/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Salmonella/isolamento & purificação , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/metabolismo , Proteínas de Bactérias/imunologia , Carne/microbiologia , Leite/microbiologia , Salmonella/genética , Sensibilidade e EspecificidadeRESUMO
Vaccines have become fundamental in the control and elimination of Glässer Disease, a systemic disease of pigs caused by Haemophilus parasuis. The classic vaccines available for prevention of this infection were developed without a robust knowledge about host immunological mechanisms. In this study, we demonstrated the presence of cross-reactive epitopes on both the N-lobe and C-lobe of variants of transferrin binding protein B (TbpBs) expressed on the surface of 6 virulent serovars of H. parasuis. Antibodies against TbpB-derived antigens were capable of increasing the phagocytic capacity of neutrophils and were also capable of blocking porcine transferrin from binding to TbpB. Surprisingly, none of the pig or mice antisera from animals immunized with TbpB-derived antigens mixed with Montanide IMS 2215 VG PR adjuvant were able to activate the classical complement pathway (CCP). In contrast, antisera from mice immunized with TbpB-derived antigens adjuvanted with Freund's adjuvants or Montanide Gel 01 were able to activate the CCP and kill H. parasuis. Our results demonstrate that the type of adjuvant can modulate the functional response induced by TbpB-derived antigens. Based on these results, we propose that a properly formulated TbpB-based vaccine may elicit a functional protective antibody response with broad cross-reactivity against heterologous strains of H. parasuis.
Assuntos
Anticorpos Antibacterianos/metabolismo , Epitopos/imunologia , Vacinas Anti-Haemophilus/imunologia , Haemophilus parasuis/imunologia , Proteína B de Ligação a Transferrina/química , Adjuvantes Imunológicos/administração & dosagem , Animais , Reações Cruzadas , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/prevenção & controle , Infecções por Haemophilus/veterinária , Vacinas Anti-Haemophilus/administração & dosagem , Haemophilus parasuis/patogenicidade , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Transferrina/metabolismo , Proteína B de Ligação a Transferrina/genética , Proteína B de Ligação a Transferrina/imunologia , VirulênciaRESUMO
Bioelectrochemical sensing of Mycobacterium tuberculosis through electro-immunosensors is a promising technique to detect relevant analytes. In general, immunosensors require the formation of organic assemblies by the adsorption of molecular constituents. Moreover, they depend on the correct immobilization of the bio-recognition element in the biosensor. These procedures cannot be easily monitored without the use of invasive methods. In this work, an impedance analysis technique was used, as a non-invasive method, to measure and differentiate the manufacturing stages of the sensors. Biomicrosystems were fabricated through physical vapor deposition (PVD) of 80 nm Au nanolayers on 35 µm copper surfaces. Later, the surface was modified through thiolation methods generating a self-assembled-monolayer (SAM) with 20 mM 4-aminothiophenol (4-ATP) on which a polyclonal antibody (pAb) was covalently attached. Using impedance analysis, every step of the electro-immunosensor fabrication protocol was characterized using 40 independent replicas. Results showed that, compared to the negative controls, distilled water, and 0.5 µg/mL HSA, a maximum variation of 171% between each replica was achieved when compared to samples containing 0.5 µg/mL of ESAT-6 M. tuberculosis immunodominant protein. Therefore, this development validates a non-invasive method to electrically monitor the assembly process of electro-immunosensors and a tool for its further measure for detection of relevant antigens.
Assuntos
Anticorpos Antibacterianos/análise , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Microtecnologia/métodos , Mycobacterium tuberculosis/isolamento & purificação , Compostos de Anilina/química , Anticorpos Antibacterianos/metabolismo , Compostos de Sulfidrila/químicaRESUMO
Accurately identifying Mycobacterium bovis-infected cattle is critical for bovine tuberculosis prevention and control. One method for identifying infected cattle is an ELISA developed by IDEXX laboratories, which detects antibodies to two M. bovis proteins, MPB70 and MPB83. The assay's sensitivity varies by geographic region, with sensitivities of 77%, 45%, and 9% in bovine serum samples from the United Kingdom (n = 126), the United States (n = 146), and Mexico (n = 128), respectively. We hypothesized that geographically-biased sequence variation in mpb70 and mpb83, or in the genes that regulate their expression (sigK and rskA), may explain these differing sensitivities. This hypothesis was tested by comparing the sequences of these four genes in 455 M. bovis strains isolated from cattle in the aforementioned countries. For each gene, a single, common sequence was identified in most genomes of the M. bovis strains collected in all three countries. Twelve of the 455 strains were isolated from infected cattle for which the IDEXX ELISA was also performed. Five of the seven ELISA-positive genomes and three of the five ELISA-negative genomes contained the most common sequence of all four genes. Thus, sequence variation in mpb70, mpb83, sigK, and rskA does not explain the geographic disparities in IDEXX ELISA sensitivity.
Assuntos
Proteínas de Bactérias/genética , Mycobacterium bovis/classificação , Mycobacterium bovis/genética , Análise de Sequência de DNA/métodos , Tuberculose Bovina/diagnóstico , Animais , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Bovinos , Ensaio de Imunoadsorção Enzimática , Variação Genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , México , Mycobacterium bovis/imunologia , Sensibilidade e Especificidade , Tuberculose Bovina/imunologia , Reino Unido , Estados UnidosRESUMO
Salmonella Enteritidis is the main cause of foodborne salmonellosis worldwide. The limited effectiveness of current interventions against this pathogen has been the main incentive to develop new methods for the efficient control of this infection. To investigate the use of DNA vaccines against S. Enteritidis in humans, immune responses stimulated by two plasmids containing the genes designated SEN1002, located in the pathogenicity island SPI-19 and encoding a Hcp protein involved in transport mechanisms, and SEN1395, located in the genomic island ΦSE14 and encoding a protein of a new superfamily of lysozymes, were evaluated. Humoral and cellular responses following intranasal immunization of two groups of BALB/c mice with the plasmids pV1002 and pV1395 plus adjuvant were evaluated and it was observed that the IgG2a/IgG1 ratios were sixfold higher than control groups. Both plasmids stimulated specific secretory IgA production. Increased proliferation of lymphocytes and IFN-γ production were detected in both experimental groups. DNA-vaccinated mice developed protective immunity against a virulent strain of S. Enteritidis, with nearly 2 logs of protection level compared to the negative control values in the spleen. Therefore, DNA vaccines are efficient at stimulating cellular and humoral immune responses at systemic and mucosal levels.
Assuntos
Imunidade nas Mucosas/imunologia , Salmonelose Animal/imunologia , Vacinas contra Salmonella/imunologia , Vacinação , Adjuvantes Imunológicos/farmacologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/metabolismo , Feminino , Imunidade nas Mucosas/efeitos dos fármacos , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Fases de Leitura Aberta , Salmonelose Animal/microbiologia , Salmonella enteritidis , Vacinas de DNA/imunologiaRESUMO
Escherichia coli are bacteria that must be controlled in the food industry and the hospital sector. Magnetoelastic biosensors offer the promise of rapid identification of these and other harmful antigens. In this work, strips of amorphous Metglas 2826MB3 were cut to size (5 mm × 1 mm) with a microdicing saw and were then coated with thin layers of Cr and Au, as verified by Rutherford backscattering spectroscopy (RBS). Several sensor surfaces were studied: 1) as-cast strip, wheel side; 2) as-cast strip, free surface; and 3) thinned and polished surface. A layer of cystamine was applied to the Au-covered magnetoelastic substrate, forming a self-assembledmonolayer (SAM), followed by antibodies, using a modified Hermanson protocol. The cystamine layer growth was verified by Fourier transform infrared spectroscopy (FTIR) and scanning electronmicroscopy (SEM). The biosensors were exposed to solutions of bacteria and the resonant frequency of the sensors was measured with an impedance analyzer for times up to 100 min. Reductions in the resonant frequency, corresponding to bacteria capture, were measured after optimizing the signal amplitude. For times up to 40 min, high capture rates were observed and thereafter saturation occurred. Saturation values of the frequency shifts were compared with the number of bacteria observed on the sensor using fluorescence microscopy. Parameters associated with capture kinetics were studied for different sensor surfaces. The rough surfaces were found to show a faster response, while the thinned and polished sensors showed the largest frequency shift.
Assuntos
Técnicas Biossensoriais/métodos , Escherichia coli/isolamento & purificação , Anticorpos Antibacterianos/metabolismo , Técnicas Biossensoriais/instrumentação , Cistamina , Elasticidade , Escherichia coli/metabolismo , Imãs , Propriedades de SuperfícieRESUMO
The purpose of this study was to compare histopathological changes in the lungs of chickens infected with avian pathogenic (APEC) and avian fecal (A(fecal)) Escherichia coli strains, and to analyze how the interaction of the bacteria with avian macrophages relates to the outcome of the infection. Chickens were infected intratracheally with three APEC strains, MT78, IMT5155, and UEL17, and one non-pathogenic A(fecal) strain, IMT5104. The pathogenicity of the strains was assessed by isolating bacteria from lungs, kidneys, and spleens at 24 h post-infection (p.i.). Lungs were examined for histopathological changes at 12, 18, and 24 h p.i. Serial lung sections were stained with hematoxylin and eosin (HE), terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) for detection of apoptotic cells, and an anti-O2 antibody for detection of MT78 and IMT5155. UEL17 and IMT5104 did not cause systemic infections and the extents of lung colonization were two orders of magnitude lower than for the septicemic strains MT78 and IMT5155, yet all four strains caused the same extent of inflammation in the lungs. The inflammation was localized; there were some congested areas next to unaffected areas. Only the inflamed regions became labeled with anti-O2 antibody. TUNEL labeling revealed the presence of apoptotic cells at 12 h p.i in the inflamed regions only, and before any necrotic foci could be seen. The TUNEL-positive cells were very likely dying heterophils, as evidenced by the purulent inflammation. Some of the dying cells observed in avian lungs in situ may also be macrophages, since all four avian E. coli induced caspase 3/7 activation in monolayers of HD11 avian macrophages. In summary, both pathogenic and non-pathogenic fecal strains of avian E. coli produce focal infections in the avian lung, and these are accompanied by inflammation and cell death in the infected areas.